Cell cycle specific, differentially tagged ribosomal proteins to measure phase specific transcriptomes from asynchronously cycling cells

Asynchronously cycling cells pose a challenge to the accurate characterization of phase-specific gene expression. Current strategies, including RNAseq, survey the steady state gene expression across the cell cycle and are inherently limited by their inability to resolve dynamic gene regulatory networks. Single cell RNAseq (scRNAseq) can identify different cell cycle transcriptomes if enough cycling cells are present, however some cells are not amenable to scRNAseq. Therefore, we merged two powerful strategies, the CDT1 and GMNN degrons used in Fluorescent Ubiquitination-based Cell Cycle Indicator (FUCCI) cell cycle sensors and the ribosomal protein epitope tagging used in RiboTrap/Tag technologies to isolate cell cycle phase-specific mRNA for sequencing. The resulting cell cycle dependent, tagged ribosomal proteins (ccTaggedRP) were differentially expressed during the cell cycle, had similar subcellular locations as endogenous ribosomal proteins, incorporated into ribosomes and polysomes, and facilitated the recovery of cell cycle phase-specific RNA for sequencing. ccTaggedRP has broad applications to investigate phase-specific gene expression in complex cell populations.


ccTaggedRPs stably expressed in HEK293-T cells were incorporated into endogenous ribosomes and polysomes
To control for the copy number of recombinant DNA, we created the stable expression of ccTaggedRP through Flp-In insertion of a single copy of ccTaggedRP into HEK293-T cells (Fig. 2A).Immunofluorescent staining of Flag and HA identified distinct cells expressing Flag-RPLa10-hCdt alone, Flag-RPL10a-hCdt and HA RPL10a-hGem, and HA-RPLa10-hGem alone (Fig. 2b).The majority of cells were only positive for Flag-RPLa10-hCdt alone, consistent with G1 phase of the cell cycle.
To evaluate the incorporation of recombinant ccTaggedRPs into ribosomes, we performed sucrose gradient centrifugation of lysates prepared from HEK-293 T cells stably expressing ccTaggedRPs.Cell lysates were fractionated on a 10% to 50% sucrose gradient while measuring the absorbance at 260 nm to detect ribosomal subunits (40S and 60S), in addition to 80S ribosomes, and translating ribosomes (polysomes) (Fig. 3A).Immunoblots of fractions identified the presence of recombinant Flag-RPL10a-hCdt and HA-RPL10a-hGem in the 60S large ribosomal subunits, 80S ribosomes, and polysomes, with a distribution similar to RPL10a.(Fig. 3B-D).Negative staining electron microscopy of fraction #5 demonstrated that 80S ribosomes were enriched in this fraction as expected (Fig. 3E).The findings support that Flag-RPL10a-hCdt and HA-RPL10a-hGem expressed from recombinant ccTaggedRP were incorporated into ribosomes and polysomes.

ccTaggedRP expression changes during cell cycle synchronization
The HEK293-T Flp-In cells expressing ccTaggedRP were synchronized via serum starvation, which is known to increase the percentage of cells in G 0 /G 1 as starvation persists.In accordance with expectations, the expression of Flag-RPL10a-hCdt recombinant protein was progressively increased with starvation, suggesting an enrichment in the Flag-RPL10a-hCdt construct in G 0 /G 1 while the expression of HA-RPL10a-hGem recombinant protein progressively decreased, suggesting an enrichment in HA-RPL10a-hGem construct in G 2 /M.(Supplemental Fig. 1A, B).Specifically, compared to serum-fed HEK293-T Flp-In cells, HA-RPL10a-hGem expression decreased by 82% after 48 h of serum starvation.(Supplemental Fig. 1C).
To further validate the cell cycle dependence of Flag-RPL10a-hCdt and HA-RPL10a-hGem expression, HEK293-T Flp-In cells expressing ccTaggedRP were synchronized by treatment with Lovastatin, Hydroxyurea or Nocodazole and enrichment in G 1 , S, and G 2 /M were confirmed by fluorescent activated cell sorting (FACS) (Fig. 4A-C and Supplemental Fig. 2).Cells treated with Lovastatin were 80.7% G 1 , 0.5% S, and 18.8% G 2 /M.Hydroxyurea were 25.1% G 1 , 57.7% S, and 17.2% G 2 /M.Cell treated with Nocodazole were 19.5% G 1 , 5.2% S, and 75.3% G 2 /M (Fig. 4B).Based on the FACS data and predicted ideal behavior of the degrons with Flag expressed in G1 and S and HA expressed in S and G2/M, the relative changes in Flag and HA expression were estimated (Fig. 4C).Western blotting of cells treated with Lovastatin, Hydroxyurea, or Nocodazole showed reciprocal changes in the expression of Flag-RPL10a-hCdt and HA-RPL10a-hGem (Fig. 4d).Flag-RPL10a-hCdt was increased and decreased in expression in G1 and G2M phases, respectively.HA-RPL10a-hCdt was decreased and increased in expression in G1 and G2M phases, respectively.

ccTaggedRP identifies genes expressed during G 2 -M in asynchronously cycling cells
Asynchronous HEK293-T Flp-In cells stably expressing ccTaggedRP were cultured to 60% confluency prior to non-denaturing lysis.Recombinant Flag-RPL10a-hCdt and HA-RPL10a-hGem proteins were then immunoprecipitated (IP) using HA or Flag antibodies, and their associated RNAs were recovered for bulk RNA-sequencing (Fig. 5A).Principal component analysis (PCA) displayed distinct clustering of the HA-and Flag-IP samples (Fig. 5B).This method resolved 2,125 differentially expressed genes with an adjusted p-value ≤ 0.05 between the   www.nature.com/scientificreports/HA-and Flag-IP RNA samples (Fig. 5C and Supplemental Table 1).Transcripts enriched in the HA-IPs were associated with regulation of the mitotic cell cycle and G 2 -M transition of the mitotic cell cycle while transcripts enriched in the Flag-IPs were associated with the negative regulation of both the mitotic cell cycle and the G 2 -M transition of the mitotic cell cycle (Fig. 5D).Additionally, Gene Set Enrichment (GSE) Analysis identified enrichment of G 2 -M cell cycle and checkpoint pathways in the HA-IPs (Fig. 5E).Furthermore, expression of G 2 -M genes was significantly increased in the HA-IPs compared to the Flag-IPs (Fig. 5F).

scRNAseq of HEK293-T cells identifies populations of G 1 -S and G 2 -M
To further validate ccTaggedRP, scRNAseq data for HEK293-T cells were sourced from 10× Genomics for comparison of transcripts expressed during G 1 /S and G 2 /M.Cells within the dataset were assigned to either "G 1 /S" or "G 2 /M" identities based on gene expression (Fig. 6A and Supplemental Figs. 3 and 4, and Supplemental Table 2).G 1 /S cells exhibited increased expression of MCM5, a protein important in DNA synthesis and upregulated in G 1 /S while G 2 /M cells exhibited increased expression of CCNB1, a protein important in cell cycle division and upregulated in G 2 /M (Fig. 6B, C).In this dataset, G 1 /S cells and G 2 /M cells accounted for 62.5% and 37.5% of the total number of cells, respectively (Fig. 6D).Differential expression analysis between the G 1 /S and G 2 /M cells revealed 530 differentially expressed genes with an adjusted p-value ≤ 0.05 (Fig. 6E).Hypergeometric testing of the differentially expressed genes indicated an enrichment in DNA replication processes and mitotic cell cycle processes for cells in G 1 /S and G 2 /M respectively (Fig. 6F, G).Furthermore, G 2 /M cells displayed increased expression of G 2 -M genes compared to G 1 /S cells (Fig. 6H).www.nature.com/scientificreports/

Comparison of genes expressed in G 2 -M during asynchronous cycling from ccTaggedRP and scRNAseq
To investigate the differences between the two methodologies, functionalization of shared and method-specific differentially expressed genes was performed.Comparison of differentially expressed genes from the ccTaggedRP method and scRNAseq found 2019 genes specific to the ccTaggedRP method, 423 genes specific to the scRNAseq method, and 107 genes shared between the two (Supplemental Fig. 5A).Furthermore, hypergeometric testing of the shared differentially expressed genes indicated enrichment of genes associated with mitotic cell cycle processes (Supplemental Fig. 5B).

Discussion
We sought to develop an extension of the RiboTrap/RiboTag technologies that would facilitate the characterization of transcribed genes during distinct cell cycle phases of asynchronously cycling cells.RiboTrap and Ribo-Tag use an EGFP-tagged RPL10a ribosomal protein subunit or HA-tagged RPL22 ribosomal protein subunit, respectively, for translating ribosome affinity purification (TRAP) of mRNA populations and characterization of gene expression 2-4,24-27 .Both methods have provided new insights regarding the gene expression of specific cell types in complex tissues.For example, RiboTrap has been used to investigate the differential gene expression of excitatory neurons and interneuron subtypes 28 , interstitial/pericytes, myeloid cells, nephron cells, and endothelial cells 29 while Ribotag has been used to characterize gene expression in neurons 3 , microglia 30 , oocyte maturation 31 , and cardiomyocyte and endothelial cells 4 in diverse disease states.Notably, the gene expression is obtained from bulk RNA sequencing of pooled mRNAs, meaning that the total pool of expressed transcripts dilutes the cell cycle phase-specific transcripts and modifies interpretation.To overcome this limitation, we describe the firstgeneration ccTaggedRP technology based on merging aspects of FUCCI and RiboTag methods.FUCCI employs www.nature.com/scientificreports/degrons that target proteins for degradation during the G 1 /S or G 2 /M phases of the cell cycle 22,23 .By creating ccTaggedRP, which contains different epitope-tagged RPL10a proteins with either hCdt1 or hGem1 degrons, the mRNAs expressed during cell cycle phases can be enriched by TRAP.The initial polypeptide is bicistronic and cleaved into distinct tagged RPL10a chimeric proteins whose subsequent abundance is dictated by the phase of the cell cycle via hCdt1-and hGem-mediated degradation.Additionally, the targeted insertion of a single copy of ccTaggedRP in HEK293-T-Flp-In cells avoided the effects of multiple copy insertion or insertion into a transcriptionally silenced region of the genome, which could adversely affect the expression of ccTaggedRP.3xFlagTag-RPL10a-hCdt(30-120) and 3xHA-Tag-RPL10a-hGeminin were differentially expressed during G 1 /S and G 2 /M based on synchronization of different phases of the cycle using inhibitors, had similar subcellular locations as endogenous ribosomal proteins, were incorporated into the 60S ribosomal subunit, 80S ribosomes, and polysomes, and facilitated TRAP of mRNAs expressed during G 1 /S and G 2 /M.Bulk RNA sequencing of mRNAs associated with the immunoprecipitation of 3xHA-Tag-RPL10a-hGeminin identified transcripts enriched in the regulation of the mitotic cell cycle and G 2 -M transition of the mitotic cell cycle.Conversely, the immunoprecipitation of 3xFlagTag-RPL10a-hCdt(30-120) identified transcripts enriched in the negative regulation of the mitotic cell cycle and the G 2 -M transition of the mitotic cell cycle.Collectively, our data suggests the predominance of the HA recombinant protein and Flag recombinant protein in the G 2 /M and G 1 /S phases of the cell cycle, respectively.
Next, we compared the results of TRAP using ccTaggedRP with scRNAseq results of HEK293-T cells obtained by 10× Genomics.Cells from the scRNAseq were assigned to either G 1 /S or G 2 /M phases based on their expression profiles.The differential expression between the cell cycle clusters was then compared to those obtained by ccTaggedRP.A comparison of differentially expressed genes from the ccTaggedRP method and scRNAseq found 2019 genes specific to the ccTaggedRP method, 423 genes specific to the scRNAseq method, and 107 genes shared between the two methods.Evaulation of the shared differential gene expression confirmed similar gene expression using the two methods; however, the ccTaggedRP approach resolved nearly four-fold more differentially expressed genes between conditions.Moreover, the transcript enrichment from the ccTaggedRP method was similar if not greater than that of the scRNAseq method.Additionally, the significantly lower cost of bulk RNAseq and its consequent opportunity for multiple biological replicates further bolster the potential of ccTaggedRP as an alternative to scRNAseq for resolving cell cycle-specific gene expression.
There are, however, limitations to the ccTaggedRP technology.First, the exchange rates of RPLs into and out of ribosomes are likely different for each RPL and could affect the efficiency of the ccTaggedRP technology (Supplemental Fig. 6).For example, RPLs with slower exchange rates may not degrade efficiently during cell cycle phases and influence the interpretation of TRAP data.Therefore, future ccTaggedRPs that use different RPLs will require careful characterization, given the dynamic properties of RPLs.This problem may be further exacerbated for rapidly cycling tissue, in which there is insufficient time for degradation of the constructs between phases.FUCCI constructs encode fluorescently labeled degrons and may be more amenable to degradation compared to Flag-RPL10a-hCdt and HA-RPL10a-hGem that are incorporated into ribosomes, potentially slowing their degradation.However, HA-RPL10a-hGem demonstrated more pronounced expression changes in response to the cell cycle.As presented in this study, we were able to resolve cell cycle-specific gene expression in a highly replicative embryonic kidney epithelial cell line, suggesting that the kinetics of the construct are sufficiently fast to support analyses in most contexts.Second, the heterogeneity of ribosomes could influence the interpretation of TRAP data 32 .Third, we acknowledge the presence of lower molecular weight HA and FLAG recombinant proteins, which are likely products of protein degradation.This is to be expected as each of the recombinant proteins have C-terminal degrons.Importantly, despite the presence of these degradation products, cell cycle-specific gene expression was still able to be discerned.Finally, we tested the technology in asynchronous cells in culture, a scenario that provided adequate signal detection.Translating the ccTaggedRP technology to a transgenic mouse for in vivo experiments may be challenging.
Despite these limitations, the ccTaggedRP method is promising and has great potential to identify cell cycle phase-specific gene expression in complex tissues.Moreover, it can be combined with other technologies to create even more powerful approaches.The ccTaggedRP construct could be inserted downstream of tissue-specific promoters to resolve cell cycle-specific gene expression in specific tissues.This may be particularly useful due to the high degree of contamination observed in other purification methods.Furthermore, in tissues with low replicative capacities, this methodology affords the ability to pool multiple biological samples to characterize these sparse populations.Alternatively, the methods could be adapted to create chimeras of epitope-tagged Argonaut proteins containing hCdt1 and hGem to survey the expression of microRNAs expressed during G 1 /S and G 2 /M in asynchronous cycling cells in complex tissues.

Methods details
The Flp-In HEK293-T cells stably expressing ccTaggedRP cell line and plasmid are available upon reasonable request to the corresponding author.The CAG-ccTaggedRP plasmid will be deposited into Addgene.

Negative stain EM
For negative stain electron microscopy, a 10 µL sample corresponding to the 80S ribosome peak (fraction 5) was applied directly after fractionation to a glow discharged 400-mesh grid (EMS, CF400-Cu-50) and incubated for 5 min at room temperature.The grid was stained with 2% uranyl acetate twice for 1 min each.Excess fluid was blotted away with Whatman filter paper after each incubation period (Cytiva, 1001-090).Images were collected on an FEI F20 electron microscope operated at 120 kV and equipped with a TVIPS TemCam XF416 camera.
Images were taken at a magnification of 62,000×, and a pixel size of 0.17 nm.

Flow cytometry
Cells were incubated with 10 µM EdU for 1.5 h prior to harvest.Following harvest, cells were incubated with fixable LIVE/DEAD stain (CAT#: L34992, ThermoFischer) for 20 min in the dark and then washed with PBS.Following staining, cells were fixed with 4% paraformaldehyde (PFA) for 20 min and then were washed with 1% BSA.Cells were permeabilized and underwent the Click-it reaction according to manufacturer's instructions (CAT#: C10633, ThermoFischer).Cells were then washed with permeabilization buffer and stained with FxCycle Violet (CAT#: F10347, ThermoFischer) for 1 h in the dark.Data was obtained with a BD LSR Fortessa and analyzed with OMIQ (Dotmatics).Gating strategy is illustrated in Supplemental Fig. 2. were added, and the resulting mixture was incubated overnight with rocking at 4 °C.Magnetic bead-target complexes were collected with a magnetic stand, and the remaining solution was aspirated.Samples were washed twice with IP lysis buffer and resuspended in RW1 buffer.RNA was then isolated with the RNeasy mini kit (CAT#: 74104, Qiagen) according to the manufacturer's instructions.Isolated mRNA samples were quantified and submitted to Genewiz for further processing and sequencing.

RNA sequencing analyses
cDNA library preparation with adapter ligation, and sequencing using an Illumina® HiSeq® system were performed by Genewiz, Inc. Reads underwent quality control, trimming, alignment, and quantification according to Genewiz standard workflows.Sequence reads were trimmed to remove possible adapter sequences and nucleotides with poor quality using Trimmomatic v.0.36.The trimmed reads were mapped to the Homo sapiens GRCh38 reference genome available on ENSEMBL using the STAR aligner v.2.5.2b.The STAR aligner is a splice aligner that detects splice junctions and incorporates them to help align the entire read sequences.BAM files were generated as a result of this step.Below are the statistics of mapping the reads to the reference genome.Genes with less than ten reads in at least three samples (the minimum sample size for a group) were excluded for subsequent analysis.Differential expression analysis was performed using R and the DEseq2 package.Functionalization was performed using ClusterProfiler package in R and GSEA software.

scRNAseq analysis
A scRNAseq dataset of cultured HEK293-T cells was sourced from 10× Genomics (10× Genomics).Sequencing yielded 737,280 cells with an average of 66.58 reads per cell and 19.45 genes per cell.Poor quality cells were removed, using the following inclusion criteria: cells with greater than 2000 genes, greater than 7500 reads, and less than 7.5% mitochondrial DNA.Cells were assigned a "G 2 /M" or "G 1 /S" identity using the CellCycleScoring algorithm in Seurat and previously published data sets (supplemental Fig. 7) 34,35 .Cells unable to be assigned confidently to either identity were discarded before downstream analysis.After filtering, 2447 cells remained with an average of 15,651.61reads per cell and 3485.21genes per cell.Data was normalized and scaled with the Seurat package in R. UMAP dimensionality reduction was performed with 11 principal components and a resolution of 0.5.Differential expression analysis based on the cell cycle cluster was performed using the Seurat package in R. Functionalization was performed using clusterProfiler package in R.

Quantification and statistical analysis
Shapiro Wilk test was performed on continuous variables to evaluate the data distribution.Normally-distributed data and non-normally distributed data were then analyzed for statistical significance by Student t test and Mann-Whitney U test, respectively.Statistical significance for RNAseq and scRNAseq data was determined

Figure 1 .
Figure 1.ccTaggedRPs are differentially expressed during the cell cycle and have similar subcellular locations as endogenous ribosomal proteins.(A) Schematic of the CAG-3xFlagTag-RPL10a-hCdt(30-120)-T2A-3xHA-Tag-RPL10a-hGeminin(1-110) construct and cleavage at the T2A site.(B) Schematic of the predicted change in 3xFlagTag-RPL10a-hCdt(30-120) and 3xHA-Tag-RPL10a-hGeminin(1-110) during the cell cycle.(C) Immunoblots of asynchronously cycling HEK293-T cells transiently transfected with empty plasmid, ccTaggedRP, or not transfected.Blots were probed with antibodies against Flag Tag, HA Tag, or mRPL10a.Bands corresponding to Flag recombinant protein, HA recombinant protein, and endogenous RPL10a isoforms are denoted by arrows.See Supplemental Fig. 6 for uncut blots.(D) Immunofluorescence of asynchronously cycling HEK293-T cells transiently transfected with ccTaggedRP stained for RPL10a and Flag Tag.Cells expressing ccTaggedRP are denoted by arrows.Cells not expressing ccTaggedRP are denoted by asterisks.DAPI was used to stain for nuclear DNA.DAPI is blue, RPL10a is red, and FLAG is green.Scale bars are included in the lower right corner and correspond to 10 μm.

Figure 4 .
Figure 4.The expression of Flag-RPL10a-hCdt and HA-RPL10a-hGem is dependent on the phase of the cell cycle.(A) FACS quantitation of HEK293-T-Flp-In cells stably expressing ccTaggedRPs in G 1 , S, and G 2 /M treated with after synchronization with Lovastatin, Hydroxyurea or Nocodazole.The percentage of cells in G 1 , S, and G 2 /M under each treatment is shown.B525/50-A EdU AF488 represents EdU intensity and V450/50-A DAPI represents DNA content.These measurements were used to gate cells in G 1 , S, and G 2 M. (B) Quantitation of G 1 , S, and G 2 /M of cells treated with Lovastatin, Hydroxyurea, or Nocodazole.Values are mean ± SEM.P-values were calculated by ANOVA with Tukey test for multiple comparisons.N = 4 plates per condition.(C) Predicted relative expression of Flag and HA based on the results shown in panel B. (D) Western blots of Flag-RPL10a-hCdt (top), HA-RPL10a-hGem (middle), and GAPDH (bottom) corresponding to the cells treated with Lovastatin, Hydroxyurea, or Nocodazole and analyzed by FACS in panel A. See Supplemental Fig. 10 for uncropped blots.

Figure 5 .
Figure 5. ccTaggedRP identifies genes expressed during G 2 -M in asynchronously cycling cells.(A) Schematic of the immunoprecipitation of 3xFlagTag-RPL10a-hCdt(30-120) and 3xHA-Tag-RPL10a-hGeminin(1-110) for the purification and sequencing of RNA from asynchronously cycling HEK293-T-Flp-In cells expressing ccTaggedRP.(B) Principal component analysis (PCA) plot of RNA sequencing of mRNAs from immunoprecipitated Flag recombinant protein (red dots) and HA recombinant protein (blue dots).(C) Volcano plot of RNA sequencing of mRNAs from immunoprecipitated Flag recombinant protein and HA recombinant protein.The gray line denotes an adjusted p-value of 0.05.Genes with increased and decreased expression are denoted by red and blue dots, respectively.(D) Waterfall plot of the 1090 genes enriched in the 3xFlagTag-RPL10a-hCdt(30-120) and 1035 genes enriched in the 3xHA-Tag-RPL10a-hGeminin(1-110) immunoprecipitated samples.GO Terms are denoted with adjusted p-values shown.(E) Gene set enrichment (GSE) analysis of RNAseq of mRNAs from immunoprecipitated Flag recombinant protein and HA recombinant protein.Adjusted p-vales are shown.(F) Heat map of the expression of the top 50 G 2 -M genes from mRNAs from immunoprecipitated Flag recombinant protein and HA recombinant protein.

Figure 6 .
Figure 6.scRNAseq of HEK293-T cells identifies populations of G 1 -S and G 2 -M.(A) UMAP of cells assigned to G 1 /S and G 2 /M identity in HEK293-T cell culture.(B,C) UMAP of MCM5 and CCNB1 expression.(D) Percentage of total cells assigned to G 1 /S and G 2 /M identities.(E) Volcano plot of differential gene expression between G 1 /S and G 2 /M clusters.(F,G) Gene Ontology results via hypergeometric testing of genes enriched in the G 1 /S cluster and G 2 /M cluster.(H) Heat map of the average expression for the top 40 G 2 /M genes between the G 1 /S and G 2 /M clusters. https://doi.org/10.1038/s41598-024-52085-5